鞭毛蛋白毒素导致的黑松超微结构病理学变化

【目的】以松材线虫、致病细菌荧光假单胞菌及其鞭毛蛋白毒素、非致病细菌缓慢葡萄球菌等材料对3年生黑松进行接种试验,通过其样枝切片的超微结构病理学变化,了解致病细菌毒素在松萎蔫病中的作用。【方法】分别以野生松材线虫悬液、荧光假单胞菌菌株GcM5-1A无细胞滤液、无菌松材线虫+荧光假单胞菌菌株GcM5-1A、荧光假单胞菌菌株GcM5-1A的鞭毛蛋白毒素Flg、无菌松材线虫+缓慢葡萄球菌菌株AM2C、无菌松材线虫悬液接种黑松,以健康松树为对照。对于发病的处理,在接种后病程的第2或第3阶段取样; 对于未发病的处理,则在接种11 d后取样。取样时,在距离接种点不同位置进行,然后制备透射电镜样品,观察这些切片的超微结构变化。【结果】野生松材线虫、GcM5-1A无细胞滤液、无菌松材线虫+GcM5-1A、GcM5-1A鞭毛蛋白毒素等4种处理下黑松树体组织的超微结构均遭受严重破坏,病理学变化趋势一致。无菌松材线虫、无菌松材线虫+AM2C等两种处理下树体组织基本未遭受破坏,超微结构观察结果与健康松树的基本一致。GcM5-1A无细胞滤液与野生松材线虫的处理中,在靠近接种点处,前者处理下切片的超微结构受害比后者严重; 在远离接种点处,前者受害反而比后者轻。通过GcM5-1A无细胞滤液与野生松材线虫接种观察结果比较,发现松材线虫在树体内的移动加速了致病菌的扩散,导致毒素的增产和扩散,加速了松树的死亡。【结论】在接种处理的第2阶段是松萎蔫病发展的关键阶段。无菌松材线虫不能致死松树,而松材线虫携带的致病细菌产生的毒素是致死松树的真正原因,验证了松萎蔫病是松材线虫与其携带致病细菌的复合侵染导致病害的假说,而松材线虫在树体内的移动加速了致病菌的扩散,最终导致松树加速死亡。

Pathological changes in ultrastructure of the Pinus thunbergii due to flagellin toxin

【Objective】 We aimed to determine the role of bacterial toxin in pine wilt disease by examining the changes in ultrastructure of 3-year-old Pinus thunbergii branches inoculated with different materials. 【Method】The inoculation materials were suspension of Bursaphelencus xylophilus, non-cell culturing liquid of the pathogenic bacteria GcM5-1A, a mixture of aseptic B. xylophilus + the pathogenic bacteria GcM5-1A, flagellin toxin of the pathogenic bacteria GcM5-1A, aseptic B. xylophilus + non-pathogenic bacteria Staphylococcus lentus AM2C, and suspension of aseptic B. xylophilus. Healthy P. thunbergii was used as control. For the diseased pines, samples were taken from different locations at the second or third stage after inoculation. For the non-diseased pines, samples were collected after inoculating for 11 days. The samples were sliced into thin sections and ultrastructural changes were observed with a transmission electron microscope(TEM).【Result】The ultrastructures of examined tissues were severely damaged in four treatment groups, namely wild B. xylophilus, non-cell culturing liquid of the pathogenic bacteria GcM5-1A, the mixture of aseptic B. xylophilus + the pathogenic bacteria GcM5-1A, and flagellin toxin of the pathogenic bacteria GcM5-1. The tendency of their pathological variations was consistent. However, in the treatment of non-cell culturing liquid of the pathogenic bacteria GcM5, the ultrastructures were more severely damaged in the samples close to the inoculation point than in the samples taken far away from the inoculation point. In wild B. xylophilus treatment, the results were reversed. Notably, the tissues and ultrastructure showed almost no damage in aseptic B. xylophilus + non-pathogenic bacterial S. lentus AM2C and suspension of the aseptic B. xylophilus treatments, and the ultrastructure in these two treatments were similar to those in healthy pine.【Conclusion】 The results indicate that the second stage of pine wilt disease is a critical period. The toxins produced by the pathogenic bacteria carried by B. xylophilus result in pine death, rather than the aseptic B. xylophilus itself. Our results support the hypothesis that pine wilt disease is caused by both the nematode and its associated pathogenic bacteria. Comparison of wild B. xylophilus and non-cell culturing liquid of the pathogenic bacteria GcM5-1A treatments showed that the movement of PWN in the pine tree increased the rate of pathogenic bacteria dispersal, which in turn resulted in acceleration of toxin production and diffusion, hastening the death of pines.